Early Developmental Characteristics of Digestive System of Micropterus salmoides Larvae During the First Feeding and Artificial Formula Feed Adaptation

Micropterus salmoides is an economically important cultured carnivorous fish in China. In recent years, owing to the development and wide application of artificial formula feed, M. salmoides production has rapidly increased and reached 470 000 tons in 2019. However, a low survival rate of M. salmoides larvae is observed during the first feeding and artificial formula feed adaptation.In this study, to better understand the artificial formula feed adaptation of M. salmoides larvae, the developmental characteristics of the digestive tract and digestive gland of the fish larvae from 2~30 dph were observed and described using histological sections and scanning electron microscopy (SEM). Moreover, the digestive tract (stomach and intestine) characteristics of certain dead larval fish during the first feeding and the transformation of artificial formula feed were investigated.For histological analysis, the larval fish (including stomach, intestine, pyloric cecum, liver, and pancreas tissues) were dehydrated with an alcohol gradient (70%, 80%, 90%, and 100%), embedded in paraffin, cut into 5 μm sections, and stained with standard hematoxylin and eosin.For SEM analysis, the stomach, pyloric cecum, and intestine of 30 dph larval fish were fixed in 2.5% glutaraldehyde solution for 12 h. Then, the tissues were fixed in 1% osmium solution for 2 h, dehydrated with gradient alcohol (70%~100%), soaked in tert-butyl alcohol for 2 h, dried with a lyophilizer, and plated with gold by ion sputtering. Finally, the images were captured using a HITACHIX-650 scanning electron microscope.At a water temperature of (23±1)℃, the larval yolk sac and oil drop gradually decreased at 0~4 dph, the digestive tract was initially differentiated, and heartbeat, blood circulation, mouth crack, esophagus, and anus were observed at 4 dph. Larval fish were in the endogenous nutrition period at this stage. In 4~ 6 dph larval fish, the esophagus, stomach, intestine, liver, and pancreas regions gradually formed, the digestive tube opened to the outside initially, and yolk sac and oil drop significantly decreased and then completely disappeared, indicating that the larval fish entered the endo-exotrophic period. At this stage, sufficient Artemia salina should be provided to induce the larval fish to open their mouth and perform first feeding. In 10~16 dph larval fish, the stomach, pylorus caecum, and intestine were closely arranged. From the surface of the intestinal cavity, the mucosa, submucosa, muscular, and serosa layers were successively presented. The size of the mucosa columnar epithelial cells and the number of goblet cells clearly increased. The masses of hepatocytes and pancreatic cells significantly increased, and a vacuole structure appeared in the hepatocytes. The pancreatic cells were arranged closely, and the blood cells and pancreatic ducts were distributed. At this stage, larval fish entered the exogenous nutrition period. From this stage onward, larval fish are better able to transform feeding artificial formula feed step by step. In 20~24 dph larval fish, the stomach gland was well developed, and the stomach wall thickness and number of stomach glands further increased. Furthermore, the mucosal folds and connective tissue of the submucosa were further differentiated, and the longitudinal mucosal folds increased and curled. In addition, secondary mucosal folds in the intestine gradually appeared and the number of mucosal folds in the pyloric cecum increased. Fatty accumulation and secretory granules were observed in the liver and pancreas, respectively, indicating that the larval hepatopancreas was similar to that of adult fish. The digestive system of larval fish was completely developed at this stage, and the larval fish had the ability to transform and adapt to artificial formula feed.No significant differences were observed in body length and body weight of M. salmoides larvae (P > 0.05) during the adaptation to artificial formula feed at 12, 16, and 20 dph. However, the survival rate of 12 dph larval fish (45.59%) was lower than that of 16 (60.60%) and 20 dph larval fish (69.83%). In addition, the body length, body weight, and survival rate of M. salmoides larvae were positively correlated with the time point of feeding artificial formula feed.SEM results showed abundant polygonal reticular mucosal folds in the gastric epidermis of 30-day-old larval fish, and there were dense secretion pores between the gastric pits. Mucosa folds with fixed shapes were observed on the inner surface of the intestine, and mucous cells, secretory pores, and secretory granules were clearly observed between the mucosal folds. Interestingly, the inner surface structure of the pyloric cecum was similar to that of the intestine. A difference was the mucosa folds without a fixed shape on the inner surface of the pyloric caecum. These mucosal folds and secretory pores are important for food digestion, absorption, and excretion, indicating that the larval fish are old enough to adapt to the artificial formula feed at this point.During the first feeding and artificial formula feed adaptation, the stomach and intestine of the larval fish were incompletely developed and accompanied by tissue damage in the dead individuals. For example, the stomach and intestinal cavity were significantly shriveled, the inner epidermis did not have the molding mucous membrane fold and goblet cells, and the mucous membrane layer was cracked or absent. These results indicate that larval fish overfeed and fail to effectively digest, absorb, and expel nutrients, which ultimately results in death associated with nutritional deficiencies, intestinal blockages, and inflammation.During the process of adaptation to artificial formula feed for M. salmoides, the development of larval digestive system was investigated. The digestive system of larval fish at 4~6 dph gradually differentiated and was still in the mixed nutrition period, and sufficient A. salina should be provided to induce the larval fish to open their mouths and perform the first feeding. At 6~16 dph, the digestive system gradually developed and larval fish entered the exogenous nutrition period, and sufficient food could be provided. At 16~20 dph, the digestive system of the larval fish completely developed, and this stage is the optimal time to switch to artificial formula feed. Our study provides basic data for feeding condition optimization of M. salmoides larvae.

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